MRI contrast agents that affect water proton relaxation lack sensitivity or specificity for molecular imaging of disease. Specific extracellular targets (e.g., ions, metabolites) can be imaged with MRI probes which have exchangeable protons. The chemical exchange saturation transfer (CEST) technique detects exchange between bulk water protons and -NHx or -OH protons in diamagnetic molecules or protons of an inner sphere of bound water that is shifted by the paramagnetic core of a lanthanide III (Ln3+) ion. Although paramagnetic CEST or PARACEST agents - especially derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA4-) - have great translational potential, quantitative molecular imaging is limited by contributions to the CEST effect from unmeasured influences of the agent's concentration and local environment (i.e., temperature and pH). We propose a new class of DOTA ligands containing Ln3+ that will allow measurement of these parameters while still retaining the CEST effect for an extracellular target. It is well known that paramagnetic complexes of 1,4,7,10-tetraazacyclododecane, primarily used as MRS shift agents, have several non- exchangeable protons that are easily detectable for agent concentration assessment and furthermore these signals are very sensitive to temperature and pH. Thus biosensor imaging of redundant deviation in shifts (BIRDS) of non-exchangeable protons (in <L voxels) can provide absolute measurements of temperature, pH, and agent concentration in the interstitial space of rat brain. To improve CEST quantification of DOTA- tetraamides, which are good PARACEST agents and biologically favored, we will incorporate properties of BIRDS into them. The new class of DOTA-tetraamides will contain both non-exchangeable and exchangeable protons (i.e., BIRDS and CEST properties) in the same probe. By exploiting C2 symmetry, one pair of ligating arms will feature BIRDS for quantitative evaluation of temperature, pH, and agent concentration, whereas another pair of ligating arms will attribute CEST for assessing the variations of an ion or a metabolite in the extracellular milieu. First, we will synthesize and characterize variants of DOTA-tetraamides which contain several non-exchangeable protons, in the form of -CH3 moieties, to enhance BIRDS properties. The -CH3 moieties surrounding the Ln3+ are designed for high BIRDS sensitivity to allow ~1 <L voxels in vivo - which is comparable to microSPECT and microPET methods - while at the same time provide sufficient chemical shift redundancy for simultaneous temperature and pH determination. Next, we will add Zn2+-, Ca2+-, and glucose- specific CEST characteristics onto prototypical multivalent DOTA-tetraamides. CEST properties will depend on each agent possessing a very large chemical shift separation between bound and bulk water and bound water lifetime of the appropriate range such that saturation transfer can enhance the CEST effect. Finally, we will conduct in vivo rat brain studies with some of the new multivalent agents that are most kinetically stable, possess low overall charge, and/or have low molecular weight. Since all new agents will be built on the DOTA framework, we expect some of them to have translational prospects. PUBLIC HEALTH RELEVANCE: Molecular imaging of extracellular targets with MRI is possible by detecting water-exchangeable protons in paramagnetic macrocyclics with chemical exchange saturation transfer (CEST). However CEST is limited by unknown influences of the agent's concentration and local environment (i.e., temperature and pH). Since MRS shift agents contain non-exchangeable protons for appraisal of these unmeasured parameters with biosensor imaging of redundant deviation in shifts (BIRDS), the new class of multivalent paramagnetic agents will contain both BIRDS and CEST properties for quantitative molecular imaging.